MOLECULAR CHARACTERIZATION OF CLINICAL ISOLATES...

MOLECULAR CHARACTERIZATION OF CLINICAL ISOLATES OF

ENTEROPATHOGENIC ESCHERICHIA COLI FROM MIRI SARAWAK

IRENE LAH

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2004



By

IRENE LAH

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

September 2004

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the

requirements for the degree of Master of Science



By

IRENE LAH

September 2004

Chairman: Professor Son Radu, Ph.D

Faculty: Food Science and Technology

A total of thirty two strains of clinical enteropathogenic Escherichia coli (EPEC) isolated from

Hospital Miri, Sarawak were examined and further characterized by various molecular

techniques. These techniques include the plasmid profiling, antimicrobial resistance, resistance

and virulence genes detection by multiplex PCR, RAPD, ERIC and PFGE genomic

fingerprinting. All the strains studied were found to exhibit multiple antibiotics resistance

patterns to twelve antibiotics [penicillin (100%), teicoplanin (100%), vancomycin (100%),

bacitrasin (97%), methicillin (97%), erythromycin (69%), ampicillin (63%), cephalothin (47%),

streptomycin (25%), chloramphenicol (16%), kanamycin (6%) and nalidixic acid (3%)] used.

Thirteen EPEC isolates were shown to encode ampicillin resistance by means of the blaTEM gene

respectively, and none of the EPEC isolates showed the presence of the sipB/C, cmlA/tetR and

blaPSE-1 genes. The plasmid profiles obtained ranged in size from 1.8 MDa to 57 MDa. Two

types of specific primer encoding the Shiga-like Toxin gene, the SLTII (584 bp) gene and SLTI

(348 bp) were utilized in the multiplex PCR assay. Analysis carried out demonstrated that all

were positive for the presence of the SLTII and SLTI gene. Two EPEC isolates analysed by PCR

were confirmed to be the O157:H7 serogroup as determined by agglutination tests with specific

antisera. Three 50% G+C contents 10-mer random primers, the Gen 1-50-02 (5’-

CCAAACTGCT-3’), Gen 1-50-08 (5’-GAGATGACGA-3’), and Gen1-50-09 (5’-

TCGCTATCTC-3’) were chosen after screening through ten random primers. In PFGE

technique carried out, two kinds of restriction enzymes, the SpeI (5’-A CTAGT-3’) and XbaI (5’-

T CTAGA-3’) were used to check for the in-situ DNA digestion pattern due to their inherit

advantages of the short sequence of these enzymes. Both the RAPD polymorphism pattern and

PFGE profile obtained showed a significant discriminatory fingerprinting among the 32 isolates

under studied. A respective dendrogram was constructed from the binary data matrix obtained

from the RAPD, ERIC and PFGE fingerprints to compare the diversity relationship among the

32 isolates. All the dendrograms were constructed utilizing the RAPDistance software package

based on the data retrieved from the presence or absence of banding pattern. All the three

molecular techniques of RAPD-, ERIC-, and PFGE genotyping showed a significant correlation

whereby the first 16 and the second 16 strains of EPEC used in this study showed a closer

relationship in the respective cluster groups as shown in the constructed dendrograms. From the

overall results obtained both the RAPD and ERIC analysis showed greater discriminatory power

compared to the other phenotypic and molecular characterization techniques used in this study.

Our results demonstrate that the antimicrobial resistance,presence of resistance and virulence

genes, plasmid profiling, multiplex PCR, RAPD-PCR fingerprinting, ERIC and PFGE profiling

methods are useful as a suitable analysis tools for a rapid and reliable molecular typing and

identification of EPEC.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi

keperluan untuk ijazah Master Sains

PENCIRIAN SECARA MOLEKUL ISOLASI KLINIKAL ENTEROPATHOGENIC

ESCHERICHIA COLI DARI MIRI, SARAWAK

Oleh

IRENE LAH

September 2004

Pengerusi: Profesor Son Radu, Ph.D

Fakulti: Sains Makanan dan Teknologi

Sejumlah tiga puluh dua strain klinikal enteropathogenic Escherichia coli (EPEC) yang

diisolasikan dari Hospital Miri, Sarawak telah diuji dan dikenalpasti dengan beberapa teknik

molekul. Teknik-teknik ini merangkumi penganalisaan profil plasmid, kerintangan antibiotik,

pengesanan gen kerintangan dan gen virulence menggunakan multiplek PCR, genomik

fingerprint RAPD, ERIC dan PFGE. Kesemua strain yang digunakan di dalam kajian ini didapati

mempamerkan kepelbagaian corak antibiotik terhadap dua belas jenis antibiotik [penicillin

(100%), teicoplanin (100%), vancomycin (100%), bacitrasin (97%), methicillin (97%),

erythromycin (69%), ampicillin (63%), cephalothin (47%), streptomycin (25%),

chloramphenicol (16%), kanamycin (6%) and nalidixic acid (3%)] yang digunakan. Tiga belas

isolat EPEC enkod kerintangan ampicillin yang bermaksud gen blaTEM dan tiada isolat EPEC

yang menunjukkan kehadiran gen sipB/C, cmlA/tetR dan blaPSE-1. Profil plasmid yang diperolehi

menunjukkan julat saiz antara 1.8 MDa ke 57 MDa. Dua jenis primer spesifik mengkod gen

“Shiga-like Toxin”, iaitu SLTII (584 bp) dan SLTI (348 bp) telah digunakan di dalam assai

multiplek PCR. Analisis yang dijalankan mendapati bahawa kesemua strain menunjukkan

kehadiran gen SLTII dan SLTI. Dua EPEC strain telah disahkan sebagai O157:H7 melalui

multiplek PCR dan ujikaji agglutination dengan spesifik antisera. Tiga primer rawak (10-mer)

yang mengandungi kandungan G+C sebanyak 50%, iaitu primer Gen 1-50-02 (5’-

CCAAACTGCT-3’), Gen 1-50-08 (5’-GAGATGACGA-3’), dan Gen1-50-09 (5’-

TCGCTATCTC-3’) telah dipilih selepas diuji secara rawak dengan 10 primer. Dalam teknik

PFGE, dua jenis enzim pemotong, iaitu SpeI (5’-A CTAGT-3’) dan XbaI (5’-T CTAGA-3’)

telah digunakan untuk diuji kepada corak. Kedua-dua corak polymorphism RAPD dan profile

PFGE yang diperolehi menunjukkan diskriminatori signifikan fingerprint antara 32 isolat dalam

kajian. Dendrogram masing-masing yang dibina daripada data binari matrik yang diperolehi dari

fingerprint RAPD, ERIC dan PFGE untuk dibandingkan dengan kepelbagaian hubungan diantara

32 isolat. Kesemua dendrogram yang dibina menggunakan RAPDistance software package

sesuai dengan data yang dicari daripada kehadiran dan ketidak hadiran corak band. Kesemua tiga

teknik molekul iaitu genotipikan RAPD, ERIC dan PFGE menunjukkan korelasi signifikan

dimana 16 strain yang pertama dan 16 strain yang kedua isolat EPEc yang digunakan di dalam

kajian ini menunjukkan hungan yang rapat antara satu sama lain bagi setiap kumpulan cluster

yang ditunjukkan di dalam dendrogram yang telah dibina. Daripada keselurahan keputusan yang

diperolehi, kedua-dua penganalisis RAPD dan ERIC menunjukkan kuasa diskriminatori yang

terbaik berbanding dengan fenotip dan pengenalpastian teknik molekul yang lain yang digunakan

di dalam kajian ini. Keputusan menunjukkan bahawa kerintangan antibiotik dan gen, profil

plasmid, multiplek PCR, fingerprint RAPD PCR, kaedah profil ERIC dan PFGE berfaedah

sebagai analisis yang sesuai untuk rapid dan jenis molekul dan identifikasi EPEC.

LIST OF TABLES

Table Page

3.1: Primer pairs utilized in the multiplex PCR. 43

3.2: The EPEC isolates used in this study. 48

4.1: Primer pairs utilized in the multiplex PCR. 57

4.2: Resistance percentage of EPEC isolates. 61

4.3: Antibiotic resistance patterns of 32 EPEC isolates. 62

4.4: Antibiotic resistance and PCR product of 32 EPEC isolates. 64

5.1: Correlation between antibiotic resistance patterns and

plasmid profiles of 32 EPEC isolates. 76

6.1: Nei-Li similarity between EPEC isolates

(RAPDistance Software Version 4.0) 91

7.1: The nucleotide sequence of ERIC primer gene pair. 98





LIST OF FIGURES

Figure Page

1: General structure of integrons. The arrows show the direction

of transcription. The location and orientation of different

promoters are shown. The sequence GTTRRRY is the integron’s

crossover point for integration of gene cassettes. The 5’-CS

and 3’-CS oligonucleotides are specific to the end 5’ and 3’

conserved segments, respectively. They were used as probes for

colony hybridization and as primers for PCR analysis of integrons.

One inserted cassette is shown, with its associated 59-base element

(37) indicated by the black bar. 25

2: Schematic diagram of the PCR amplification process. 33

3.1: The representative of multiplex PCR results (for the SLTI, SLTII

and FLICH genes) obtained for EPEC. Lane M: 100 bp DNA ladder

size marker; lanes 1 to 16: represent 16 EPEC strains no. 1-16 and

lane 17 represent isolate of Escherichia coli O157:H7 with ID number

ECEDL933 (ATCC control positive strain) respectively. 47

3.2: The representative of multiplex PCR results (for the SLTI, SLTII

and FLICH genes) obtained for EPEC. Lane M: 100 bp DNA ladder

size marker; lanes 17 to 32: represent 16 EPEC strains no. 17-32 and

lane 33 represent isolate of Escherichia coli O157:H7 with ID number

ECEDL933 (ATCC control positive strain) respectively. 47

4.1: Pie Chart showing percentage resistance of EPEC isolates towards the

total number of antibiotics with antibiotic resistance patterns group. 65

4.2: The representative of PCR results (for the blaTEM gene) obtained

for EPEC. Lanes M: 100 bp DNA ladder size marker; and

lane 1-16: EPEC strains. 66

4.3: The representative of PCR results (for the blaTEM gene) obtained

for EPEC. Lanes M: 100 bp DNA ladder size marker; and


5.1: Pie chart showing the percentage of EPEC isolates which harbor

different number of plasmid with different plasmid profiles. 78

5.2: A representative photograph of the agarose (0.7%) gel electrophoresis

of plasmid DNA profiles detected in some of the isolates of EPEC.

Lanes: M, ECV517 size reference plasmid; lanes 1 to 13: EPEC strains. 79

5.3: A representative photograph of the agarose (0.7%) gel electrophoresis

of plasmid DNA profiles detected in some of the isolates of EPEC.

Lanes: M, ECV517 size reference plasmid; lanes 14 to 32: EPEC strains. 79

6.1: RAPD fingerprinting profile obtained using primer Gen 1-50-02

(5’– CCAAACTGCT–3’) for the first 16 strains of EPEC.

Lane M: Molecular mass size marker of 1 kb DNA ladder;



(5’– CCAAACTGCT–3’) for the second 16 strains of EPEC.




(5’– GAGATGACGA–3’) for the first 16 strains of EPEC.




(5’– GAGATGACGA–3’) for the second 16 strains of EPEC.




(5’–TCGCTATCTC–3’) for the first 16 strains of EPEC.




(5’–TCGCTATCTC–3’) for the first 16 strains of EPEC.



6.7: Dendrogram of EPEC isolates generated by UPGMA clustering and tree

building NJTREE program (RAPDistance Software Version 4.0). 92

7.1: Figure showing a representative ERIC fingerprinting obtained for the

first 16 isolates of EPEC. Lane M, 1 kb DNA ladder for molecular

size marker; lanes 1-16: EPEC isolates numbered 1-16. 100

7.2: Figure showing a representative ERIC fingerprinting obtained for the

second 16 isolates of EPEC. Lane M, 1 kb DNA ladder for molecular

size marker; lanes 17-32: EPEC isolates numbered 17-32. 100

7.3: Dendrogram of EPEC isolates generated by UPGMA clustering and tree


8.1: The PFGE profiles for the 12 isolates of EPEC digested with enzyme

XbaI. Lane M: PFGE lambda DNA marker; lanes 1-12: EPEC strains. 111






SpeI. Lane M: PFGE lambda DNA marker; lanes 1-12: EPEC strains. 112





8.7 Dendrogram of EPEC isolates generated by UPGMA clustering and tree


LIST OF ABBREVIATIONS

A adenine or adenosine

AP-PCR arbitrarily primed-polymerase chain reaction

ATCC American type culture collection

ATP adenosine triphosphate

Am ampicillin

B bacitracin

bp basepair

BSA bovine serum albumin

C chloramphenicol

Car carbenicillin

Cf cephalothin

ccc covalently closed circular

CN gentamicin

cm centimetre

Da dalton (the unit of molecular mass)

dATP Deoxyadenosine triphosphate

dCTP Deoxycytosine triphosphate

dGTP Deoxyguanosine triphosphate

dH2O Distilled water

DI discriminatory index

DNA Deoxyribonucleic acid

dTTP Deoxythymidine triphosphate

E erythromycin

EAEC enteroaggregative Escherichia coli

EAF EPEC adherence factor

E. coli Escherichia coli

e.g. For example

EDTA Ethylenediamine tetraacetic acid

EHEC enterohemorrhagic Escherichia coli

EIEC enteroinvasive Escherichia coli

EPEC enteropathogenic Escherichia coli

ETEC enterotoxigenic Escherichia coli

EtBr ethidium bromide

gm gram

g gravity

G Guanine

GM gentamicin

GTP Guanosine triphosphate

H2O Water

HCl Hydrochloric acid

i.e. that is

ID Identification number

K kanamycin

KAc potassium acetate

kb Kilobase pair (number of bases in thousands)

Kda kiloDalton

kg kilogram

l litre

LB Luria-Bertani

M Molar, or molarity, moles of solute per liter of slution

mA miliamphere

MAR Multiple Antibiotic Resistance

MDa megadalton

Met methicillin

mg miligram

MHA mueller Hinton agar

min Minutes

ml Mililiter

mm millimeter

mM Millimolar

µg Microgram

µl Microliter

mol mole

Nal nalidixic acid

NaCl Sodium Chloride

NaOH Sodium hydroxide

ng Nanogram

P penicillin

% Percent

R resistant

RAPD Random Amplified Polymorphic DNA

RFLP Restriction fragment length polymorphism

RNA ribonucleic acid

Rnase ribonuclease

rpm revolution per minute

S sensitive

S streptomycin

sdH2O Sterile distilled water

SDS sodium dodecyl sulphate

TAE Tris acetate EDTA electrophoresis buffer

Taq Thermus aquaticus DNA (polymerase)

TBE Tris borate EDTA electrophoresis buffer

TE tris-EDTA

TEC teicoplanin

Tris tris (hydroxymethyl) methylamine

UV ultraviolet

V Volts

Van vancomycin

w weight

oC degree Celsius

ACKNOWLEDGEMENTS

My deepest gratitude goes to my supervisor, Professor Dr. Son Radu for trusting me and

giving me the opportunity to do my MSc. I treasured and appreciate his patience, guidance,

advices and encouragement through out my study years. I learn a lot from him.

My appreciation also goes to Associate Professor Dr. Raha Abdul Rahim and Dr.

Clemente Michael Wong Vui Ling for their support and cooperation. They have been very

helpful.

Not forgetting my family (dad, mum, Amie, Flo, Urei, Unen, Abo’ Uk, and Puyang) for

their unending love, prayers and encouragement. Words could not express my gratitude to all of

you. Special thanks goes to all my aunts, uncles, grandpa, grandma and cousins for their love and

prayers.

Also, I thank my lab friends (Wai Ling, Les, Gwen, Kqueen, Sam, Jurin, Kak Zaleha, Ibu

Endang, Sushil, John Lawrence, Yousr, Kak Zila, Yin Sze, Bell, Liha, Tung, and Daniel) for

helping me in my work and patiently teaching me so many things. Thank you for your

friendship, it means a lot to me.

Also I would like to express thanks to all my housemates (Chris, Thy, Sera, Emmy, Cath,

Gina, Arish, Ah Lai and Charlyn), our pastor (Pr. Kenny Tham and family; Pr. Jung and family),

our cell group members(Mim, Mine, Elly, Emma, and Sue) and brothers and sisters in Christ SIB

Serdang for their support, help and prayers. I am so bless to have all of you by my side….. All

glory, honor, praise and thanksgiving be unto the Lord.

Last but not least, my most heartfelt appreciation goes to Chris Gala Innue (my one and

only sweetheart) – thanks for your encouragement and prayers as well as mentally support.

CHAPTER I

GENERAL INTRODUCTION

Escherichia coli is the predominant facultative anaerobic in the human colonic flora. It

usually remains harmlessly confined to the intestinal lumen; however, in the debilitated or

immunosuppressed host, or when gastrointestinal barriers are violated, even E. coli strains of

normal flora can cause infection. Three general clinical syndromes result from infection with

pathogenic E. coli strain are (i) urinary tract infection: (ii) sepsis/meningitis; and (iii)

enteric/diarrhoeal disease (Nataro and Kaper, 1998). Moreover, even the most robust members of

our species may be susceptible to infection by one of several highly adapted E.coli clones which

together have evolved the ability to cause a broad spectrum of human diseases. Infections due to

pathogenic E. coli maybe limited to the mucosal surfaces or can disseminate throughout the

body. Three general paradigms have been described by which E. coli may cause diarrhea are (i)

enterotoxin production (ETEC and EAEC), (ii) invasion (EIEC), and/or (iii) intimate adherence

with membrane signalling (EPEC and EHEC). However, the interaction of the organisms

with

the intestinal mucosa is specific for each category. The versatility of the E. coli genome is

conferred mainly by two genetic configurations: virulence-related plasmids and chromosomal

pathogenicity islands. All six categories of diarrheagenic E. coli have been shown to carry at

least one virulence-related property upon a plasmid. EIEC, EHEC, EAEC,

and EPEC strains

typically harbor highly conserved plasmid families, each encoding multiple virulence factors

(Hales et al., 1992; Nataro and Kaper, 1987; Wood et al., 1986). McDaniel and Kaper have

shown recently that the chromosomal virulence genes of EPEC and EHEC are organized as a

cluster referred to as a pathogenicity island (McDaniel et al., 1995; McDaniel and Kaper, 1997).

Such islands have been described for uropathogenic E. coli strains (Donnenberg and Welch,

1996) and systemic E. coli strains (Bloch and Rode, 1996) as well and may represent a common

way in which the genomes of pathogenic and nonpathogenic E. coli strains diverge genetically.

Fecal-oral and food borne transmission of E.coli are well documented (Nataro and Kaper,

1998). As with other diarrheagenic E. coli, transmission of enteropathogenic E. coli (EPEC) is

fecal-oral, with contaminated hands, contaminated food, or contaminated fomites serving as

vehicles. In adults outbreak, waterborne and foodborne transmissions have been reported, but no

particular type of food has been implicated as more likely to serve as a source of infection

(Levine and Edelman, 1984). The most notable feature of type epidemiology of disease due to

EPEC is the striking age distribution seen in persons infected with this pathogen. EPEC infection

is primarily a disease of younger than 2 years. Illness caused by EPEC is often clinically acute

severe diarrhea. However, EPEC can cause diarrhea in an adult if the bacterial inoculums is high

enough (Nataro and Kaper, 1998).

Pathogenic human and animal E. coli resistant to many classes of antimicrobial agents

have been reported worldwide (Bradford et al., 1997; Winolur et al., 2001). These multi resistant

pathogens present an important challenge to achieving effective therapy. Antimicrobial

resistance in commensal strains of E. coli, however, may also play an important role in the

ecology of resistance clinical infectious diseases. Transmission of resistance genes from

normally nonpathogenic species to other virulent organism within the animal or humans interinal

tact may be an important mechanism for acquiring clinically significant antimicrobial-resistant

organisms. E. coli may serve as an important reservoir for these transmissible resistances, since it

is clear that this organism has developed a number of elaborate mechanisms for acquiring and

disseminating plasmids, transposons, phage, and other genetic determinants (Neidhardt, 1996).

The resistance phenotypes may arise from many different genetic determents and each

determinate may present specific epidemiological features. Therefore, the assessment of the

resistance situation at the genetic level would be an important asset in the understanding and

control of antimicrobial resistance in general. Antibiotic resistance genes found in nature are

organized in gene cassettes and may be included in intergrons. Intergrons may be transferred

between bacteria and higher cells at high frequencies. Disc diffusion is the method always used

in the laboratory to determine antibiotic resistance phenotypes. The resistant of isolates toward

antibiotic is shown with the clear zone formed around the antibiotic-containing disc.

The pathogenicity of EPEC is encoded by plasmids, but bacterial chromosome also

brings the effect of the pathogenesis for diarrhea. The molecular weight of plasmids can indicate

the characteristics of conjugation in transferring the resistant genes. The high copy number and

low molecular weight plasmids can indicate the efficiency in conjugation of genetic information.

Normally, plasmids bring drug-resistant genes (R plasmids) toward certain antibiotics presented

in the environment so that bacterial cells can survive in the environment.

In recent years, the use of molecular “fingerprinting” methods has become standard

practice in microbiology for evaluating the epidemiology of infectious diseases, investigating

suspected outbreaks of bacterial infection, and trying bacterial (Mickelsen, 1997). Pulsed-field

gel electrophoresis (PFGE) allows the generation of simplified chromosomal restriction fragment

patterns without having to resort to probe hybridization methods. In this method, restriction

enzymes that infrequently cut DNA are used for generating large fragments of chromosomal

DNA, which are then separated by special electrophoresis (Swaminatham and Matar, 1993).

PFGE has been applied to sub typing of several Gram-positive and Gram-negative bacteria. It is

not widely used in epidemiological surveillance and common interpretation schemes have been

published (Tenovar et al., 1997).

Another widely used method is Polymerase chain reaction (PCR). In PCR, a pair of

primers (20-40 bases) is used for selective amplification and detection of a certain DNA

sequence in a target orgasm. PCR primers have successfully been developed for all categories of

dirrheagenic E. coli. PCR can be used in both diagnosing and typing E.coli strains. Advantages

of PCR include high sensitivity, specificity and appropriate rapidity in the detection of target

DNA templates. In diagnostics, PCR is commonly used for detecting different virulence

associated genes of E. coli, such as toxin and adherence associated genes. PCR is also widely

used in sub typing by doing virulence geneproflie for different diarrheagenic E. coli strains. The

RAPD, a PCR based methods has been used as a sensitive and efficient method for

distinguishing and study of genetic relatedness among the isolates. A pair of short primers is bind

randomly to the DNA sequence and amplified into bands. The same RAPD pattern shown will

indicate the higher possibility of the isolates was derived from the same serogroups. Other

techniques such as enterobacterial repetitive intergenic consensus (ERIC) PCR (Gison et al.,

1984; Hulton et al., 2001; de Moura et al., 2001). The study conducted by Versalovic et al.

(1991) had demonstrated that the ERIC-like sequences are present in many diverse eubacterial

species, and also these sequences, can be utilized as efficient prier binding sites in the PCR

reaction to produce fingerprints of different bacterial genomes.

The latex agglutination test (Verotox-F assay, denka Seyken, Tokio, Japan) for detection

of toxins produced by Shiga Toxin-producing E. coli (STEC) has been found 100% sensitive and

100% specific in comparison with the classical Vero cell assay (Karmali et al., 1999).

Immunomagnetic separation with magnetic beads coated with antibody against E.coli O157 have

been found more sensitive than direct culture of these strains (Chapman and Siddons, 1996).

All these new molecular typing methods have allowed highly discriminant genotyping,

and are useful tools for demonstrating that isolates from different sources are identical, closely

related or not related at all (Mickelsen, 1997). The molecular biotyping techniques are now well

accepted as one of the most important and useful differentiation tool for studying the molecular

characteristics of isolates, an understanding of genetic variability in E. coli is important for

studies of the taxanomy, epidemiology and pathogenicity of this species.

Objective of Study

The objectives of this study are:

1. To detect the presence of the specific gene sequence responsible for the production of the

‘shiga like-toxin’ by multiplex-polymerase chain reaction (multiplex-PCR) technique.

2. To determine the antibiotic susceptibility patterns and plasmid profiles among EPEC

isolates.

3. To carry out DNA-based genotyping techniques for the EPEC isolated using randomly

amplified polymorphic DNA analysis (RAPD) ERIC-PRC and pulsed-field gel

electrophoresis (PFGE).

4. To compare the discriminatory power of antibiotic resistance patterns, plasmid profiles,

RAPD-, ERIC-PCR and PFGE analysis for typing the EPEC isolates.

CHAPTER II

LITERATURE REVIEW

Enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) is an important category of diarrheagenic

E. coli which has been linked to infant diarrhea in the developing world.

Once defined solely on the basis of O and H serotypes, EPEC is now

defined on the basis of

pathogenetic characteristics.

Since the 17th century, diarrhea had become the real death cause of children during

summer. So, it had been termed as “summer diarrhea” (Creighton, 1975). After one century,

Albert et al. (1995) reported again that E. coli infections peaked during dry summer months,

from February to May in Bangladesh, a tropical country. Enteropathogenic Escherichia coli

(EPEC) is a leading cause of infantile diarrhea in developing countries. In industrialized

countries, the frequency of these organisms has decreased, but they continue to be an important

cause of diarrhea (Nataro et al., 1998). The central mechanism of EPEC pathogenesis is a lesion

called attaching and effacing (A/E), which is characterized by microvilli destruction, intimate

adherence of bacteria to the intestinal epithelium, pedestal formation, and aggregation of

polarized actin and other elements of the cytoskeleton at sites of bacterial attachment. The

fluorescent actin staining test allows the identification of strains that produce A/E lesions,

through detection of aggregated actin filaments beneath the attached bacteria (Knutton et al.,

1989). Ability to produce A/E lesions has also been detected in strains of Shiga toxin-producing

E. coli (enterohemorrhagic E. coli [EHEC] and in strains of other bacterial species (Nataro et al.,

1998). The emergence and rise in frequency of atypical EPEC strains may have origins similar to

those that led to the emergence and increase in frequency of O157:H7 and other STEC serotypes

(Griffin, 1998).

Enteropathogenic Escherichia coli (EPEC) has been recognized as a common cause of

watery diarrhea in children (Moyenuddin et al., 1989; Sethi and Khuffash, 1989). A study

conducted in Malaysia (Jegathesan et al., 1975) showed that 9% of the diarrheal cases in children

under 10 years of age in Malaysia were due to EPEC. The reason(s) for the relative resistance of

adults and older children is not known, but loss of specific receptors with age is one possibility.

However, EPEC can cause diarrheal in an adult if the bacterial inoculum is high enough. The

infectious dose in naturally transmitted infection in infants is not known, but it is presumed to be

much lower than with adults (Nataro and Kaper, 1998). EPEC strains are an important cause of

disease in all settings of nosocomial outbreaks, outpatient clinics, patients admitted to hospitals,

community-based longitudinal studies, and urban and rural settings (Nataro and Kaper, 1998).

The most notable feature of the epidemiology of disease due to EPEC is the striking age

distribution seen in persons infected with this pathogen. EPEC infection is primarily a disease of

infants younger than 2 years. As reviewed by Levine and Edelman (Levine and Edelman, 1984),

numerous case-control studies in many countries have shown a strong correlation of isolation of

EPEC from infants with diarrhea compared to healthy infants. The correlation is strongest with

infants younger than 6 months. In children older than 2 years, EPEC can be isolated from healthy

and sick individuals, but a statistically significant correlation with disease is usually not found.

Another virulence property that has been associated with EPEC is the production of

verotoxin (VT), also known as Shiga-like toxin. Although strains from outbreaks usually did not

produce VT, some strains, in particular of serogroups O26 and O111, from sporadic cases of

diarrhea or hemolytic-uremic syndrome have been shown to possess this property (Smith et al.,

1990; Scotland et al., 1990; Caprioll et al., 1992; Willshaw et al., 1992; Caprioll et al., 1994)

and are currently classified as EHEC. Therefore, although the mechanism by which EPEC strains

evoke a fluid response in the intestine of the human host is still unclear, many virulence-

associated factors have been identified in this group of organisms, and some of them have been

proposed as possible markers for EPEC identification, in addition to or as replacement for the O

serogrouping (Levine et al., 1988; Knutton et al., 1989). However, recent studies suggest that not

all the virulence factors are evenly distributed in the wild-type EPEC strains circulating in

different geographical areas (Levine et al., 1988; Smith et al., 1990; Knutton et al., 1991;

Scotland et al., 1991; Morelli et al., 1994).

The Shiga toxin (stx) genes are part of the genome of temperate lambdoid phages,

which are integrated in the chromosome of the bacterial host. At present,

the ability to

produce Stx has been assigned to more than 200 E. coli serotypes, which have been isolated

from patients, healthy humans (Nataro and Kaper, 1998), animals, food (Doyle and

Schoeni, 1987), and water (Muniesa and Jofre, 1998). Stx production was observed also in

other members of the Enterobacteriaceae, including Citrobacter freundii (Schmidt et al.,

1993; Tschape et al., 1995) Enterobacter cloacae (Paton and Paton, 1996), Shigella sonnei

(Strauch et al., 2001), and Shigella dysenteriae I (Strockbine et al., 1988).

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